Phospholipid metabolism is fundamental in cells. It not only generates basic biological membranes, but also plays important roles in cellular signaling processes in nearly all tissues. In addition, many proteins, both globular and membrane bound, require specific phospholipids to fulfill their functions. Cells maintain a complicated and regulated metabolic network to synthesize a great diversity of phospholipids and degrade them in a time fashion to meet cellular requirements. Many steps of phospholipid metabolism take place on the cell membrane and are catalyzed by membrane-embedded enzymes. Their molecular mechanisms are poorly understood largely due to the paucity of structural information. In particular, how these enzymes select their substrates from the lipid membrane bilayer and carry out catalysis in a hydrophobic membrane environment is a central question still unanswered for general phospholipid metabolic mechanisms. To understand this important question, we study lipid phosphate phosphatases (LPPs) as model. LPPs, members of an intramembrane phosphatase protein family, play important roles in phospholipid synthesis and homeostasis. LPPs also catalyze dephosphorylation of several important phospholipid hormonal messengers regulating numerous phospholipids-mediated signaling processes. Based on our recent apo form crystal structure of the PgpB protein, an LPP homolog from E.coli, we proposed a novel hypothesis for the intramembrane dephosphorylation mechanism of PgpB, in which a) phospholipid substrates access an conserved intramembrane tunnel from the membrane bilayer to reach the catalytic site and b) a large conformational change of TM3 is essential for substrate binding and catalysis. To test this important hypothesis, in this project w will focus on two key aspects using a combination of biochemical, biophysical and X-ray structural approaches. 1) To demonstrate the substrate binding conformation and substrate-induced protein conformational changes, we will determine PgpB complex structures bound with a metabolism-stabilized phospholipid substrate analog or vanadate, a phosphate product analog, to gain structural details of a catalytic cycle. 2) To functionally characterize the intramembrane substrate access tunnel, we have designed several mutagenesis and crosslinking strategies to elucidate how the substrate passes through the intramembrane tunnel to reach the catalytic site. We will also explore the product release pathway using similar approaches to understand how the dephosphorylated product is delivered to the membrane bilayer after catalysis. 3) To further demonstrate the protein conformational changes, we will apply EPR and fluorescence stopped-flow approaches to catch the protein motions in response to the substrate analog binding in atomic detail in detergent solutions or in different lipid-defind nanodiscs. These structural and functional studies will not only confirm our hypothesis and reveal the catalytic mechanism of intramembrane phospholipid dephosphorylation, but also establish a structural basis to understand phospholipid metabolism in the cell membrane in general. PUBLIC HEALTH RELEVANCE: Phospholipids are essential for cellular functions. They form cell membranes and regulate cell growth, survival and development. We study lipid phosphate phosphatases, an important protein family for membrane phospholipid synthesis and hormonal phospholipid homeostasis. We aim to understand molecular mechanism underlying phospholipid dephosphorylation catalysis by this protein family. We will provide atomic structural information of a LPP protein in complex with substrate analog inhibitors and study how LPPs identify its substrates and perform novel catalysis in the cell membrane. Our proposed studies may provide important information to design therapeutic approaches for phospholipid-related cancers and cardiovascular diseases.